Summary The main mechanism of heme detoxification implemented by Plasmodium parasites is the sequestration of heme as non-toxic, crystalline hemozoin. Heme sequestration has been the most successful molecular target for antimalarial drugs. Despite many years of effort, fundamental questions regarding the mechanism of heme detoxification remain elusive. It is not clear whether the drugs inhibit crystallization by forming soluble complexes with hematin, or interact with the hemozoin surface. Other open questions relate to the detailed molecular mechanism of inhibition and the existence of specific sites on the hemozoin surface that are active in drug binding, to whether different antimalarials utilize similar or different mechanisms and whether artemisinin derivatives interfere with heme detoxification. We propose, for the first time in antimalarial research, to elucidate the molecular mechanisms of inhibition of crystal growth by antimalarials and provide atomic-level detail of the relevant active sites on hemozoin crystal surfaces. We will pursue two specific aims: 1. Establish the mechanisms of action in blocking hematin crystallization of several classes of antimalarial drugs and related compounds. 2. Provide an atomic-level view of the active sites for hematin incorporation into crystals and association of the antimalarials and monitor the dynamics of antimalarial drug association with these sites in real time. Our main method of investigation is time-resolved in situ atomic force microscopy (AFM), including atomic resolution AFM, pioneered for studies of hematin crystallization by our group. Completion of the work proposed here will guide us to additional fundamental issues of heme detoxification and its inhibition. Achieving aim 1 will allow us to rank the drugs according to their potency in crystallization inhibition and explore how the crystals respond to the increased supersaturation due to the accumulation of hematin. Achieving aim 2 will provide the basis for state-of-the-art molecular dynamics modeling (using quantum mechanical potentials and explicit solvent) to address drug-hematin interactions in solution and elucidate drug binding modes on crystal surfaces. Such modeling will evolve into a platform for rational design of new antimalarials, to be developed in collaboration with medicinal chemists and parasitologists to study parasite suppression, drug bioavailability, and efficacy.